JP2017511802A - Immunogenic liposome preparation - Google Patents

Abstract

Liposome compositions comprising liposomes and aminoalkanoic acid sulfone buffers are described and claimed. [Selection] Figure 1

Description

DESCRIPTION OF FEDERALLY SPONSORED RESEARCH Aspects of the present invention were implemented with United States government support under NIH contract number HHSN272200900008C, and the United States government may have certain rights in the invention.

  Toll-like receptor 4 (TLR4) agonists are immunogenic compounds. TLR4 agonists have been formulated in liposomes for delivery. Monophosphoryl lipid A is a known TLR4 agonist. 3-O-deacetylated monophosphoryl lipid A (MPL) is formulated in a liposome composition in a vaccine. There is a need for general improved liposomal compositions, particularly improved liposomal compositions of TLR4 agonists for administration to human subjects. A potent TLR4 agonist liposome composition with high incorporation efficiency is desired.

  The present invention relates to improved liposomes for use in pharmaceutical compositions.

  In one embodiment, the present invention provides a liposomal composition comprising a lipid, suitably a phospholipid, and an aminoalkane sulfonate buffer such as HEPES, HEPPS / EPPS, MOPS, MOBS and PIPES.

  In another suitable embodiment, the present invention provides a liposomal composition comprising a lipid, such as a phospholipid, and an aminoalkyl glucosaminide phosphate (AFP), suitably CRX-601.

  In another suitable embodiment, the present invention provides a liposomal composition comprising a lipid, AGP and an aminoalkane sulfonate buffer, wherein the lipid is suitably a phospholipid.

  In other suitable embodiments, the present invention is an improved method of producing liposomal compositions comprising: dioleoylphosphatidylcholine (generally, optionally with pharmaceutically active ingredients such as cholesterol and / or AGP) Dissolving a lipid such as “DOPC”) in an organic solvent, removing the solvent to obtain a phospholipid membrane, adding the membrane to an aminoalkanesulfone buffer, dispersing the membrane in solution, and polycarbonate A method is provided comprising continuously extruding a solution from a filter to form unilamellar liposomes. The liposomal composition can be further sterile filtered.

  These novel liposome compositions have a significantly higher incorporation efficiency of AGP, which is known to be potent and potentially reactive. Formulation of AGP liposome compositions for pharmaceutical use as described herein may result in an improved therapeutic index of the composition as compared to other formulations of agonists.

  In one embodiment, the liposome composition exhibits high incorporation of TLR4 agonists when the liposome is formed with and without cholesterol, providing advantages for the production and formulation of such liposome compositions. .

  The liposomes of the present invention are useful for both production and use of pharmaceutical compositions.

  Further embodiments are disclosed in the description, figures and claims provided herein.

CRX-601 LAL data showing incorporation efficiency determined by comparing sample slope and start time with reference to 1N (0% incorporation). CRX-601 LAL data showing incorporation efficiency determined by comparing sample slope and start time with reference to 1N (0% incorporation). LAL data showing incorporation efficiency determined by comparing sample slope and start time with reference to CRX-527 Hepes (0% incorporation).

Liposomes The term “liposomes” generally refers to monolayer or multilamellar encapsulating aqueous content (specifically 2, 3, 4, 5, 6, 7, depending on the number of lipid membranes formed. (8, 9, or 10 layers) lipid structure. Liposomes and liposome formulations are well known in the art. Lipids capable of forming liposomes include all substances having fat or fat-like properties. Lipids that can constitute lipids in liposomes are glycerides, glycerophospholipids, glycerophosphinolipids, glycerophosphonolipids, sulfolipids, sphingolipids, phospholipids, isoprenolides, steroids, stearin, sterols, alkeolipids, synthetic cationic lipids and carbohydrates It can be selected from the group comprising carbohydrate containing lipids.

  In certain embodiments of the invention, the liposome comprises a phospholipid. Phosphocholine (PC), an intermediate in the synthesis of (but not limited to) phosphatidylcholine as a suitable phospholipid; natural phospholipid derivatives: egg phosphocholine, egg phosphocholine, soy phosphocholine, hydrogenated soy phosphocholine, sphingomyelin as natural phospholipid; And synthetic phospholipid derivatives: phosphocholine (didecanoyl-L-α-phosphatidylcholine [DDPC], dilauroylphosphatidylcholine [DLPC], dimyristoylphosphatidylcholine [DMPC], dipalmitoylphosphatidylcholine [DPPC], distearoylphosphatidylcholine [DSPC], dioleoyl Phosphatidylcholine [DOPC], 1-palmitoyl, 2-oleoylphosphatidylcholine [POPC], dielide phosphatidylcholine [DEPC]), phosphoglycerol (1,2-dimyristoyl-sn-glycero-3-phosphoglycerin [DMPG], 1 , 2 -Dipalmitoyl-sn-glycero-3-phosphoglycerin [DPPG], 1,2-distearoyl-sn-glycero-3-phosphoglycerin [DSPG], 1-palmitoyl-2-oleoyl-sn-glycero-3- Phosphoglycerin [POPG]), phosphatidic acid (1,2-dimyristoyl-sn-glycero-3-phosphatidic acid [DMPA], dipalmitoyl phosphatidic acid [DPPA], distearoyl-phosphatidic acid [DSPA])), phosphoethanolamine (1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine [DMPE], 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine [DPPE], 1,2-distearoyl-sn- Glycero-3-phosphoethanolamine DSPE, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine [DOPE]), phosphoserine, polyethylene glycol [PEG] phospholipid (mPEG-phospholipid, polyglycerin-phospholipid, Functionalized phospholipids, terminal activation Phospholipids), 1,2-dioleoyl-3- (trimethylammonium) propane (DOTAP) can be mentioned. In one embodiment, the liposome comprises 1-palmitoyl-2-oleoyl-glycero-3-phosphoethanolamine. In one embodiment, highly purified phosphatidylcholine is used, which can be selected from the group comprising phosphatidylcholine (egg), hydrogenated phosphatidylcholine (egg), phosphatidylcholine (soybean) and hydrogenated phosphatidylcholine (soybean). In further embodiments, the liposome comprises phosphatidylethanolamine [POPE] or a derivative thereof, or may comprise sphingomyelin (SPNG).

  The liposome size can vary from 30 nm to about 5 μm depending on the phospholipid composition and the method used to prepare it. In certain embodiments of the invention, the liposome size ranges from 30 nm to 500 nm, and in further embodiments, from 50 nm to 200 nm, suitably less than 200 nm. Dynamic laser light scattering is a method used to measure liposome diameters well known to those skilled in the art.

  In particular, the liposomes of the present invention may comprise dioleoylphosphatidylcholine [DOPC] and sterols, particularly cholesterol.

Liposome composition A “liposome composition” is a prepared composition comprising liposomes and inclusions in the liposome, in particular, lipids forming the liposome bilayer, compounds other than lipids in the liposome bilayer, aqueous liposomes. Compounds within and associated with the contents, and compounds bound to or associated with the outer layer of the liposome. Thus, the liposomal compositions of the present invention may suitably include, but are not limited to, pharmaceutically active ingredients, vaccine antigens and adjuvants, excipients, carriers, and buffers in addition to liposomal lipids. . In preferred embodiments, such compounds are complementary to the stability or AGP incorporation efficiency of the liposome composition and / or are not significantly detrimental.

  By “liposome formulation” is meant a liposomal composition suitably formulated with other compounds for storage and / or administration to a subject as described herein.

  Accordingly, the “liposome formulation” of the present invention includes the liposome composition of the present invention, and further includes, but is not limited to, liposome compositions outside the scope of the present invention, as well as pharmaceutically active ingredients, vaccine antigens and adjuvants, and excipients. Agents, carriers and buffers can be included. In preferred embodiments, such compounds are complementary to and / or not significantly detrimental to the stability or AGP incorporation efficiency of the liposomal compositions of the invention.

Aminoalkylglucosaminide phosphate compounds
AGP is a Toll-like receptor 4 (TLR4) modulator. Toll-like receptor 4 recognizes bacterial LPS (lipopolysaccharide) and initiates an innate immune response when activated. AGP is a monosaccharide mimetic of the lipid A protein of bacterial LPS and was developed by ether and ester linkages on the “acyl chain” of the compound. Methods for making such compounds are known, for example, WO 2006/016997, US Patent Nos. 7,288,640 and 6,113,918, and WO 01, which are hereby incorporated by reference in their entirety. / 90129. Other AGP and related methods are disclosed in US Pat. No. 7,129,219, US Pat. No. 6,525,028 and US Pat. No. 6,911,434. AGPs having an ether linkage on the acyl chain used in the compositions of the present invention are known and are disclosed in WO 2006/016997, which is incorporated herein by reference in its entirety. Of particular importance are the aminoalkyl glucosaminide phosphate compounds described and illustrated in formulas (III) of paragraphs [0019] to [0021] in WO 2006/016997.

[式中、
mは0〜6であり、
nは0〜4であり、
XはO又はS、好ましくは、Oであり、
YはO又はNHであり、
ZはO又はHであり、
R₁、R₂、R₃はそれぞれ独立にC_1〜20アシル及びC_1〜20アルキルからなる群から選択され、
R₄はH又はMeであり、
R₅は独立に-H、-OH、-(C₁〜C₄)アルコキシ、-P0₃R₈R₉、-OPO₃R₈R₉、-SO₃R₈、-OSO₃R₈、-NR₈R₉、-SR₈、-CN、-NO₂、-CHO、-CO₂R₈、及びCONR₈R₉からなる群から選択され、ここでR₈及びR₉はそれぞれ独立にH及び(C₁〜C₄)アルキルから選択され、
R₆及びR₇がそれぞれ独立にH又はPO₃H₂である]
に記載された構造を有する。 The aminoalkyl glucosaminide phosphate compound used in the present invention has the following formula 1

[Where
m is 0-6,
n is 0-4,
X is O or S, preferably O,
Y is O or NH;
Z is O or H;
R ₁ , R ₂ , R ₃ are each independently selected from the group consisting of C _1-20 acyl and C _1-20 alkyl;
R ₄ is H or Me;
R ₅ is independently -H, -OH,-(C ₁ -C ₄ ) alkoxy, -P0 ₃ R ₈ R ₉ , -OPO ₃ R ₈ R ₉ , -SO ₃ R ₈ , -OSO ₃ R ₈ ,- NR ₈ R ₉ , —SR ₈ , —CN, —NO ₂ , —CHO, —CO ₂ R ₈ , and CONR ₈ R ₉ , wherein R ₈ and R ₉ are each independently H and (C ₁ ~C ₄₎ is selected from alkyl,
R ₆ and R ₇ are each independently H or PO ₃ H ₂ ]
It has the structure described in 1.

In Formula 1, the configuration of the 3 ′ stereocenter to which a normal aliphatic acyl residue (i.e., a secondary acyloxy or alkoxy residue such as R ₁ O, R ₂ O, and R ₃ O) is attached is: R or S, preferably R (named according to Cahn-IngoLd-PreLog priority rule). The configuration of the aglycone stereocenter to which R ₄ and R ₅ are bonded may be R or S. All stereoisomers, both enantiomers and diastereomers, and mixtures thereof are considered to be within the scope of the present invention.

  The number of carbon atoms between the heteroatom X and the aglycone nitrogen atom is determined by the variable “n”, which may be an integer from 0 to 4, preferably from 0 to 2.

The chain length of normal fatty acids R ₁ , R ₂ and R ₃ may be from about 6 to about 16 carbons, preferably from about 9 to about 14 carbons. The chain lengths may be the same or different. Some preferred embodiments include chain lengths where R1, R2 and R3 are 6 or 10 or 12 or 14.

Formula 1 includes L / D-seryl, -threonyl, -cysteinyl ether and ester lipid AGP, both agonists and antagonists and their homologs (n = 1-4) and various carboxylic acid biological equivalents ( That is, R ₅ is an acidic group capable of forming a salt, and the phosphate may be on the 4-position or 6-position of the glucosamine unit, and is preferably 4-position.

[式中、XはO又はSであり、YはO又はNHであり、ZはO又はHであり、R₁、R₂、R₃はそれぞれ独立にC_1〜20アシル及びC_1〜20アルキルからなる群から選択され、R₄はH又はメチルである]
の構造として記載される。 In a preferred embodiment of the invention using an AGP compound of formula 1, n is 0, R ₅ is CO ₂ H, R ₆ is PO ₃ H ₂ and R ₇ is H. This preferred AGP compound has the formula 1a

Wherein X is O or S, Y is O or NH, Z is O or H, R ₁ , R ₂ and R ₃ are each independently C _1-20 acyl and C _1-20 Selected from the group consisting of alkyl and R ₄ is H or methyl]
It is described as the structure of

In Formula 1a, the configuration of the 3 ′ stereocenter to which a normal aliphatic acyl residue (i.e., a secondary acyloxy or alkoxy residue, such as R ₁ O, R ₂ O, and R ₃ O) is attached is R or S, preferably R (named according to Cahn-IngoLd-PreLog priority rule). The configuration of the aglycone stereocenter to which R ₄ and CO ₂ H are bonded may be R or S. All stereoisomers, both enantiomers and diastereomers, and mixtures thereof are considered to be within the scope of the present invention.

  Formula 1a includes L / D-seryl, -threonyl, -cysteinyl ether or ester lipid AGP, both agonists and antagonists.

  In both Formula 1 and Formula 1a, Z is O bonded by two hydrogen atoms bonded by a double bond or a single bond respectively. That is, this compound is an ester bond when Z = Y = O, an amide bond when Z = O and Y = NH, and an ether bond when Z = H / H and Y = O. .

加えて、別の好ましい実施形態は、示される構造を有するCRX547を用いる。

さらに他の実施形態は、CRX602又はCRX526などのAGPを含み、これは、より短い第2級アシル又はアルキル鎖を有するAGPに対しての増加した安定性をもたらす。

本発明で使用するのに適した他のAGPには、CRX524及びCRX529が含まれる。 Particularly preferred compounds of formula 1 are referred to as CRX-601 and CRX-527. Its structure is described as follows:

In addition, another preferred embodiment uses CRX547 having the structure shown.

Still other embodiments include AGPs such as CRX602 or CRX526, which provide increased stability against AGPs having shorter secondary acyl or alkyl chains.

Other AGPs suitable for use with the present invention include CRX524 and CRX529.

Buffering Agent In one embodiment of the invention, the liposome composition is buffered using a zwitterionic buffering agent. Suitably the zwitterionic buffer is an aminoalkanesulfonic acid or a suitable salt. Examples of aminoalkanesulfonic acid buffers include, but are not limited to, HEPES, HEPPS / EPPS, MOPS, MOBS, and PIPES. Preferably, the buffering agent is a pharmaceutically acceptable buffer suitable for human use, such as for use with commercially available injection products. Most preferably, the buffer is HEPES. The liposomal composition can suitably comprise AGP.

  In a suitable embodiment of the invention, the liposomes are buffered using HEPES having a pH of about 7.

  In a preferred embodiment of the invention, AGP CRX-601, CRX-527 and CRX-547 are included in a liposome composition buffered using HEPES having a pH of about 7. Buffering agents can be used with appropriate amounts of saline or other excipients to achieve the desired isotonicity. In one preferred embodiment, 0.9% saline is used.

HEPESは、周知であり、市販されている(例えば、Goodら、Biochemistry1966を参照されたい)。 HEPES: CAS registration number: 7365-45-9 C ₈ H ₁₈ N ₂ O ₄ S
1-piperazineethanesulfonic acid, 4- (2-hydroxyethyl)-
HEPES is a physiological pH range of about 6 to about 8 (e.g., 6.15 to 8.35), more specifically a more useful range of about 6.8 to about 8.2, in the present case about 7 to about 8, or Zwitterionic buffers designed to be buffered at 7-8, preferably about 7 to less than 8. HEPES is usually a white crystalline powder and has the molecular formula C ₈ H ₁₈ N ₂ O ₄ S with the following structure:

HEPES is well known and commercially available (see, eg, Good et al., Biochemistry 1966).

Preparation of liposomes Standard methods for making liposomes include, but are not limited to, methods reported in Liposomes: A Practical Approach, VPTorchiLin, Volkmar Weissig Oxford University Press, 2003, and are well known in the art. .

One suitable method for making the liposomal compositions of the present invention includes AGP (e.g., CRX-601 (20 mg) and DOPC (specifically, 1,2-dioleoyl-sn-glycero-3-phosphocholine, 400 mg)) and optionally sterols (eg cholesterol (100 mg)) are dissolved in an organic phase of chloroform or tetrahydrofuran in a round bottom flask. The organic solvent is removed by evaporation on a rotary evaporator and an additional 12 hours of high pressure vacuum. To the mixed phospholipid membrane thus obtained, 10 mL of aminoalkanesulfonic acid buffer such as 10 mM HEPES buffer or 10 mM HEPES-saline buffer at pH 7.2 is added. The mixture is sonicated on a water bath (20-30 ° C.) with intermittent vortexing until all membranes along the flask wall are dispersed in the solution (30 minutes to 1.5 hours). The solution is then continuously extruded from the polycarbonate filter using a lipid mini-extruder (Lipex ^™ extruder (Northern Lipids Inc., Canada)) to form unilamellar liposomes. The liposome composition is then sterile filtered using a 0.22 μm filter and placed in a sterile depyrogen container. The average particle size of the resulting formulation as measured by dynamic light scattering was 80-120 nm and had a net negative ζ potential. The formulation exhibits final target concentrations of CRX-601 2 mg / mL, cholesterol 10 mg / mL, and total phospholipids 40 mg / mL.

  The aminoalkyl glucosaminide 4-phosphate (AGP) CRX-601 used in this work can be synthesized as previously described (Bazin, 2008 32447 / id) and can be purified by chromatography. (Up to 95% purity). CRX-601, either in the starting material or in the final product, can be quantified by standard reverse phase HPLC analysis methods.

  CRX-601 formulated in HEPES buffer (pH = 7.0) is 5 times faster than liposome hydration buffer (LHB based on phosphate, pH = 6.1). A diameter reduction was obtained. Rehydration of the CRX-601 lipid membrane in HEPES buffer required 4 times less total pressure and time to formulate liposomes compared to LHB phosphate buffer. This is a significant improvement because it saves both energy and time and places much less stress on AGP during liposome processing.

  In one embodiment, suitable ranges of components of the liposomal composition include lipids in the range of about 3-4% w / v, 1% w / v sterol, AGP in the range of 0.1-1% w / v, etc. Contains active ingredient and 10 mM aminoalkanesulfonic acid buffer. In one embodiment, the sterol is suitably present in the range of 0.5-4% w / v. Further, in one embodiment, the lipid: sterol: active ingredient ratio is about 3-4: 1: 0.1-1.

Example 1: CRX-601 formulation lipid compatibility test
Eight lipids were screened with CRX-601 in an optimal liposomal formulation of CRX-601 that provides maximum stability of the API, and in tests to find acceptable pyrogenicity and / or toxicity associated with adjuvants.

Lipex Extruder ^™ was used to prepare the formulation. pH 7.0 10 mM HEPES was selected as the hydration buffer. A liposomal formulation was prepared at a target concentration of 2 mg / mL in HEPES buffer at pH = 7.0. These lipid formulations are subject to 6-8 real-time stability at 2-8 ° C and accelerated stability at 40 ° C for 14 days, zeta potential to incorporate appearance, particle size, and aggregation, percent incorporation of CRX-601 The degradation of CRX-601 was monitored by RP-HPLC over time, along with any changes in chromogenic Limulus amebocyte lysate (LAL) to account for changes in

組込み効率を、LALデータから、CRX-601 1N参照（0%組込み）に関してサンプルの傾き及び開始時間を比較することによって決定した。以下に示すt=0のLALデータは、DOPC、DOPC Chol、DOPC DC-Chol、DOTAP、及びDOTAP CholにおいてCRX-601について、及びCRX-527 DOPCについて、良好な組込みを示した(図1及び3)。残りの製剤は、図2に示す様に乏しい組込みを示した。 Table 1 shows t = 0 (process data) for all prepared liposomes.

Integration efficiency was determined from the LAL data by comparing the slope and start time of the sample with respect to the CRX-601 1N reference (0% incorporation). The t = 0 LAL data shown below showed good integration for CRX-601 and CRX-527 DOPC in DOPC, DOPC Chol, DOPC DC-Chol, DOTAP, and DOTAP Chol (Figures 1 and 3). ). The remaining formulation showed poor incorporation as shown in FIG.

Example 2: Built-in efficiency test
To determine the effect of CRX-601 on liposome incorporation, LAL assays were performed on various liposome compositions with or without cholesterol. The data obtained (not shown) confirmed a faster LAL action indicating that DOPC and DOPC cholesterol compositions have surprisingly high levels of CRX601 incorporation. However, the results of these LAL assays were not sensitive or consistent enough to draw conclusions regarding the effect of cholesterol on incorporation.

  The rabbit pyrogenicity test was used as a surrogate evaluation criterion for CRX-601 incorporation into liposomes and as an evaluation criterion for its stability in physiological environments. This study was performed at Pacific Biolabs (Hercules, CA) according to its SOP 16E-02. The individual temperature rise from 3 rabbits per test is shown in the table below.

The data in Table 2 shows that up to 4 mg CRX-601 / ml DOPC liposome formulation prepared with or without cholesterol is non-pyrogenic up to a dose of 1000 ng / kg. This lack of pyrogenicity corresponds to a 400-fold improvement over CRX-601 (2.5 ng / kg maximum non-pyrogenic dose) and indicates greater than 99% incorporation of CRX-601 into the liposome bilayer .

The data in Table 3 shows that up to 8 mg CRX-601 / ml DOPC cholesterol liposome formulation is non-pyrogenic up to a dose of 500 ng / kg. This lack of pyrogenicity corresponds to a 200-fold improvement over CRX-601 free (maximum non-pyrogenic dose of 2.5 ng / kg) and indicates greater than 99% incorporation of CRX-601 into the liposome bilayer .

CRX-527 is an ester analog of CRX601. The data in Table 4 shows that up to 2 mg CRX-527 / ml DOPC cholesterol liposome formulation is non-pyrogenic up to a dose of 500 ng / kg. This lack of pyrogenicity indicates very high incorporation of CRX-601 into the liposome bilayer (potentially over 99%). Interestingly, unlike CRX-601, CRX-527 in the DOPC liposome formulation (ie in the absence of cholesterol) was shown to be pyrogenic at 500 ng / kg.

Good incorporation results are also shown for cationic DOTAP and DOTAP cholesterol liposomes with CRX-601.

Claims (27)

  A liposome composition comprising a liposome and an aminoalkanesulfonic acid buffer.   2. The liposome composition according to claim 1, wherein the aminoalkanesulfonic acid buffer is selected from the group comprising HEPES, HEPPS / EPPS, MOPS, MOBS and PIPES.   The liposome composition according to claim 1 or 2, wherein the aminoalkanesulfonic acid buffer is HEPES.   Lipids that make up the liposome consist of glycerides, glycerophospholipids, glycerophosphinolipids, glycerophosphonolipids, sulfolipids, sphingolipids, phospholipids, isoprenolides, steroids, stearin, sterols, alkeolipids, synthetic cationic lipids and carbohydrate-containing lipids The liposome composition according to any one of claims 1 to 3, which is selected from the group.   The liposome composition according to any one of claims 1 to 4, wherein the lipid in the liposome is a phospholipid.   The liposome composition according to any one of claims 1 to 5, wherein the lipid in the liposome is dioleoylphosphatidylcholine (DOPC).   The liposome composition according to any one of claims 1 to 6, wherein the lipid in the liposome is 1,2-dioleoyl-3- (trimethylammonium) propane (DOTAP).   The liposome composition according to any one of claims 1 to 7, further comprising sterol and in particular cholesterol.   A liposome composition comprising AGP incorporated in a liposome, wherein the liposome comprises dioleoylphosphatidylcholine (DOPC) in the absence of sterols.   The liposome composition according to any one of claims 1 to 9, wherein AGP is CRX-601.   11. The liposome composition according to any one of claims 1 to 10, wherein AGP is present in an amount greater than 10 mg / mL.   The AGP is present in an amount of less than 10 mg, less than 9 mg, less than 8 mg, less than 7 mg, less than 6 mg, less than 5 mg, less than 4 mg, less than 3 mg, less than 2 mg or less than 1 mg, but in an amount greater than 0 mg / mL. The liposome composition according to any one of the above.   13. The liposome composition according to any one of claims 1 to 12, wherein AGP is present in an amount greater than 0 mg / mL but not more than 10 mg.   14. The liposome composition according to any one of claims 1 to 13, wherein AGP is present in an amount of 30 [mu] g / mL to 6 mg / mL.   The liposome composition according to any one of claims 1 to 14, wherein the liposome is a multilamellar.   The liposome composition according to any one of claims 1 to 15, wherein the liposome is 2, 3, 4, 5, 6, 7, 8, 9, or 10 layers.   The liposome composition according to any one of claims 1 to 16, wherein the liposome is a monolayer.   18. Liposome composition according to any one of claims 1 to 17, wherein the liposome diameter ranges from 50nm to 500nm, in a further embodiment ranges from 50nm to 200nm.   19. The liposome composition according to any one of claims 1 to 18, wherein the liposome diameter is in the range of about 80 nm to 120 nm.   20. The liposome composition according to any one of claims 1 to 19, wherein the liposome structure encapsulates an aqueous content.   21. The liposome composition according to any one of claims 1 to 20, further comprising a lipid A mimetic, a TLR4 ligand, or AGP. The activity is of formula I:

[b.
cm is 0-6,
dn is 0-4,
eX is O or S, preferably O,
fY is O or NH;
gZ is O or H;
hR ₁ , R ₂ , R ₃ are each independently selected from the group consisting of C _1-20 acyl and C _1-20 alkyl;
iR ₄ is H or Me,
-H, -OH, independently jR ₅ is - (C ₁ ~C _{4) alkoxy, -P0 3 R 8 R 9,} -OP0 3 R 8 R 9, -S0 3 R 8, -OS0 3 R 8, - NR ₈ R ₉ , -SR ₈ , -CN, -N0 ₂ , -CHO, -C0 ₂ R ₈ , and CONR ₈ R ₉ , wherein R ₈ and R ₉ are each independently H and (C ₁ ~C ₄₎ is selected from alkyl,
kR ₆ and R ₇ are each independently H or PO ₃ H ₂ ]
The liposome composition according to any one of claims 1 to 21, which is an aminoalkyl glucosaminide phosphate having the structure shown in 1.   23. The liposome composition according to any one of claims 1 to 22, further comprising active (Lipid A mimetic).   Selected from the group consisting of CRX527, 601, 602, 547, 529 and 529 (TLR4 ligand) (AGP). A method for improving the production of liposome compositions comprising:
Preparing the phospholipid membrane, and adding the phospholipid file to the aminoalkanesulfone buffer. A method for producing a liposome composition comprising:
a. dissolving a lipid such as DOPC in an organic solvent (optionally with pharmaceutically active ingredients such as cholesterol and / or AGP),
b. removing the solvent to obtain a phospholipid membrane;
c. adding a phospholipid membrane to the aminoalkanesulfone buffer;
d. dispersing the membrane in the solution; and
e. A method comprising continuously extruding a solution from a polycarbonate filter to form unilamellar liposomes.   27. The method of claim 26, further comprising the step of sterile filtering the extruded liposomes.

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